Isolation,Identification And Characterization Of Salmonella Phage And Its Application In Food Control

Salmonella spp.are important foodborne pathogens,causing significant health threats and economic losses to humans every year.With the abuse of antibiotics,resistant Salmonella strains are constantly appearing.Therefore,it is urgent to develop new high-efficiency bacteriostatic agents.Phages can specifically lyse bacteria and its encoded lysin can destroy bacterial cell wall to cause lysis and death,so they have good prospect on the prevention and control of pathogenic bacteria.Therefore,both of them are increasingly used as substitutes for antibiotics.In this study,114 phages were isolated from sewage et al samples from environment by using Salmonella as the host bacteria,and 7 broad-spectrum phages were selected to study their biological characteristic;whole genome sequencing and bioinformatics analysis of the strong lytic phage T139 were analyzed,prediction of lysin associated with bacterial lysis and recombinant expression to obtain cleavage-active proteins;in addition,the antibacterial effect of strong lytic phage T139 on Salmonella in milk and beef were studied,and also its antibacterial effect on resistant Salmonella in egg,lettuce and sausage,all results laid a theoretical foundation for the development of new antibacterial agents.The main research results are listed below:（1）Isolation,purification and biological characteristics of Salmonella phages:114 strains of phage were isolated and purified by Salmonella Typhimurium,Salmonella Paratyphi B and Salmonella Choleraesuls as the host strain,and 7 phages with different plaque morphology and broad host spectrums were selected,namely T41,T135,T139,T144,P17,P22 and C22,respectively.Their biological characteristics were studied.Phage T41 belonged to Myovridae,T135,P17,P22 and C22 belonged to Sipsoviridae,T139 and T144 belonged to Podoviridae by transmission electron microscopy.The best multiplicity of infection（MOI）for 7 phages were 0.001.The optimal adsorption time of phage T41,T135,T139,T144,P17,P22 and C22 were 15 min,10 min,6 min,9 min,20min,20 min and 20 min,respectively,and the optimum adsorption rates were 91.16%,87.51%,65.86%,77.57%,90.39%,82.28%,90.70%,respectively.The latend period of phage T41,T135,T139,T144,P17,P22 and C22 were 20 min,10 min,5 min,5 min,20min,10 min and 10 min,respectively,the lytic period were 100 min,120 min and 60,65min,100 min,100 min,80 min,respectively,the average burst size were 36.76 PFU/cell,13.53 PFU/cell,54.54 PFU/cell,6 1.76 PFU/cell,25.32 PFU/cell,17.38 PFU/cell,35.83PFU/cell,respectively.All 7 phages were able to withstand high temperature conditions of50°C and the p H of 4～12.The results of the lytic curve showed that 7 phages had good lysis ability to host bacteria,among them phage T139 and T144 had the strongest lysis ability.（2）Structural protein and genomic analysis of Salmonella phage T139 and verification of lysin activity:structural proteins of phage T139 was analyzed at least 9protein bands by SDS-PAGE,and the 37 k D protein content was the largest,which was presumed to be the main capsid protein.The full-genome sequencing analysis of phage T139 showed that the phage T139 genome was 38854 bp in length with an average GC content of 49.10%,and did not contain t RNA gene.Phage T139 belonged to the T7 phage genus by the phylogenetic tree.A total of 43 ORFs were predicted,of which 23 were assigned as known functions.Functional ORFs can be divided into five major categories:nucleic acid metabolism and DNA packaging,structural protein genes except tail related proteins,cell lysis genes,tail-related proteins and unknown proteins.The p ET clone vector was selected for recombinant expression of the lysin Lys T40.The recombinant protein was successfully expressed in BL21（DE3）with a protein concentration of 280μg/m L.The lysin has good lytic activity to pretreated Salmonella Typhimurium with chloroform or EDTA,and the absorbance OD₆₀₀ decreased 0.18 and 0.31 comparing with the control group at 30 min,respectively,so the lysin had good lytic activity.（3）Application of Salmonella phage T139 in food control:the phage T139 was applied to the inhibition of Salmonella Typhimurium ATCC 13311 growing in milk and beef.For milk,there was no significant bacteriostatic effect at MOI=10 and MOI=100 at4°C,the antibacterial effect was significant after 6 h at MOI=10 and MOI=100 at 25°C,the bacterial count decreased 4.31 log₁₀ CFU/m L comparing with control at 12 h,and the sterilization efficiency exceeded 95%.For beef,the antibacterial effect was significant at MOI=100 at 4°C,the highest bacterial count decreased 0.91 log₁₀ CFU/m L comparing with control,the sterilization efficiency exceeded 70%,and the antibacterial effect was significant after 3 h at MOI=10 and MOI=100 at 25°C,the highest bacterial count decreased 1.16 log₁₀ CFU/m L comparing with control,the sterilization efficiency up to93%.The phage T139 was applied to the inhibition of resistant Salmonella Typhimurium10855 in eggs,lettuce and sausage.For egg,there was no significant antibacterial effect at MOI=1000 at 4°C,the antibacterial effect was significant at MOI=10000,and the highest bacterial count decreased 1.04 log₁₀ CFU/m L comparing with control,the sterilization efficiency exceeded 65%,the antibacterial effect was significant after 6 h at MOI=1000 at25°C,the highest bacterial count decreased 3.03 log10 CFU/m L comparing with control,and the bactericidal efficiency exceeded 95%up to 100%.For lettuce,the antibacterial effect was significant after 1 h at MOI=1000 and MOI=10000 at 4°C and 25°C,and the highest bacterial count decreased 1.92 log₁₀ CFU/m L comparing with control at 4°C,and the highest bacterial count decreased 3.17 log₁₀ CFU/m L comparing with control at 25°C,sterilization efficiency exceeds 70%up to 98%.For sausage,the antibacterial effect was significant after 1 h at MOI=1000 and MOI=10000 at 4°C and 25°C,the highest bacterial count decreased 2.04 log₁₀ CFU/m L comparing with control at 4°C,and even after 6 h at MOI=10000 at 25°C,the amount of bacteria can’t be detected,and the sterilization efficiency reached 100%.In summary,phage T139 could effectively inhibit the growth of host Salmonella and resistant Salmonella in foods,and the inhibition effect at large MOI was better than that of at small MOI,and had better inhibition effect at 25°C than 4°C.